Composition and Method for Reducing Post-Prandial Blood Glucose

ABSTRACT

A nutritional intervention composition for reducing post-prandial blood glucose levels in humans, including between about 0.1 mg and about 10 mg of a proteinase inhibitor that is administered prior to the meal. The composition is effective for treating or ameliorating the effects of hyperglycemia and Type II diabetes. The composition also is effective in combating obesity. The proteinase inhibitor is preferably isolated from plant material, such as potatoes, soy, and beans. Potato proteinase inhibitor II and soybean Bowman-Birk inhibitor are included in the group of effective proteinase inhibitors.

This application is a divisional application of U.S. patent applicationSer. No. 10/426,678, filed on Apr. 30, 2003, which claims priority toU.S. patent application Ser. No. 09/900,555, filed on Jul. 6, 2001,which issued as U.S. Pat. No. 6,767,566 on Jul. 27, 2004.

BACKGROUND OF THE INVENTION

The invention relates to compositions for reducing post-prandial bloodglucose in humans and, more specifically, to a proteinase inhibitor thatdelays gastric emptying and reduces post-prandial glycemia which may bebeneficial in combating obesity and Type II diabetes.

Regulation of body weight depends on genetic as well as physiologic andlifestyle factors that are known to influence energy balance, such asdiet, appetite control, metabolism, and physical activity (Aronne, L. J.(2001) J Clin Psychiatry 62, 13-22; Fernandez-Lopez, J. A., Remesar, X.,Foz, M. & Alemany, M. (2002) Drugs 62, 915-44). Despite measures tocombat obesity and an increased awareness of the associatedco-morbidities, the condition has become an epidemic, with nearly 60% ofAmericans classified as overweight or obese (Visscher, T. L. & Seidell,J. C. (2001) Annu Rev Public Health 22, 355-75). Since the gene pool hasnot changed, researchers believe the culprit is primarily due to acombination of environmental and lifestyle influences. A focus ondietary fat as a leading cause of obesity the last several decades hasbeen successful in reducing overall fat intake by Americans (from 40% tojust over 30% of total calories, from the 1960's to present), but hasdone little to stave the rise in obesity rates (Lichtenstein, A. H.,Kennedy, E., Barrier, P., Danford, D., Ernst, N. D., Grundy, S. M.,Leveille, G. A., Van Horn, L., Williams, C. L. & Booth, S. L. (1998)Nutr Rev 56, S3-19; discussion 519-28).

Corresponding with this profound rise in obesity incidence, a similarrise in the consumption of foods higher in processed and refinedcarbohydrates has been observed (Grundy, S. M. (1998) Am J Clin Nutr 67,563S-72S), along with an increased incidence of type II diabetes(Disdier-Flores, O. M., Rodriguez-Lugo, L. A., Perez-Perdomo, R. &Perez-Cardona, C. M. (2001) P R Health Sci J20, 123-30; Felber, J. P. &Golay, A. (2002) Int J Obes Relat Metab Disord 26 Suppl 2, S39-45).These events have led researchers to question the effect of dietary faton body fat accumulation, and suggest that dietary factors other thanfat consumption play an important role in body weight regulation(Willett, W. C. (1998) Am J Clin Nutr 67, 556S-562S; Willett, W. C.(2002) Obes Rev 3, 59-68). Evidence now exists suggesting that chronicglycemia can lead to increased fat synthesis and storage, and maycontribute significantly to the development of obesity and other chronicdiseases such as diabetes and cardiovascular disease (Jenkins, D. J.,Kendall, C. W., Augustin, L. S., Franceschi, S., Hamidi, M., Marchie,A., Jenkins, A. L. & Axelsen, M. (2002) Am J Clin Nutr 76, 266S-73S;Ludwig, D. S. (2002) JAMA 287, 2414-23; Leeds, A. R. (2002) Am J ClinNutr 76, 286S-9S). Concerns over safety and efficacy of manyanti-obesity products have limited their usefulness. Therefore,developments of natural, safe, and effective nutraceutical and/ormedications that can help treat or prevent obesity are essential tomitigate this public health crisis.

Both soybeans and potatoes are sources of proteinase inhibitors (PI's),proteins that have been hypothesized to enhance the release ofcholecystokinin (CCK), one of several gut peptides that regulate gastricemptying and satiety in humans (Liddle, R. A. (1995) Am J Physiol 269,G319-27; Beglinger, C. (1994) Ann N Y Acad Sci 713, 219-25; Beglinger,C. (2002) Curr Opin Investig Drugs 3, 587-8). Delayed gastric emptying,in turn, has been shown to result in a decreased rate of glucoseabsorption, and lower post-prandial glucose levels (Lefebvre, P. J. &Scheen, A. J. (1999) Eur J Clin Invest 29 Suppl 2, 1-6). Proteinaseinhibitor II (PI2) is a naturally occurring 21 kDa dimer and potenttrypsin and chymotrypsin inhibitor present in white potatoes (Melville,J. C. & Ryan, C. A. (1972) J Biol Chem 247, 3445-53; Bryant, J., Green,T. R., Gurusaddaiah, T. & Ryan, C. A. (1976) Biochemistry 15, 3418-24).Previous studies using large doses of highly pure PI2 demonstratedincreased CCK release and satiety in humans (Peikin, S. R., Springer, C.J., Dockray, G. J., Blundell, J. E., Hill, A. J., Calam, J. & Ryan, C.A. (1987) Gastroenterology 92, A1570; Hill, A. J., Peikin, S. R., Ryan,C. A. & Blundell, J. E. (1990) Physiol Behav 48, 241-6; Schwartz, J. G.,Guan, D., Green, G. M. & Phillips, W. T. (1994) Diabetes Care 17,255-62). In addition, oral administration of PI2 at high doses in aliquid form has been shown to reduce both post-prandial glucose andinsulin levels in humans (Schwartz, et al., supra), supporting the useof PI2 as both a promising hunger management tool and an effective agentto reduce post-prandial glycemia experienced by the body.

The development of an efficient proprietary commercial process providingan extract from potatoes containing PI2 has increased the availabilityof this compound. It was hypothesized that administration of PI2 extractas a nutraceutical ingredient in a low dose, encapsulated form, prior toa meal, might reduce post-prandial glucose levels. This could haveimportant implications for the use of PI2 as part of a diet to helpmaintain healthy blood sugar levels and reduce the propensity for weightgain.

SUMMARY OF THE INVENTION

The invention consists of a method for reducing post-prandial glycogenlevels in the blood of humans by the oral administration of a proteinaseinhibitor or a combination of proteinase inhibitors. The proteinaseinhibitor or combination is administered prior to the ingestion of ameal and reduces not only the initial rise in blood glucose following ameal (A Glucose or AG) but also the integrated area under the bloodglucose curve (AUC) following a meal. The proteinase inhibitor(s) iseffective for helping to maintain healthy blood sugar levels and fortreating persons, such as those with Type II diabetes, which haveadverse health effects due to hyperglycemia. Further, the proteinaseinhibitor(s) is expected to reduce the propensity for weight gain byreducing the glycemia experienced by the body.

Proteinase inhibitors which exhibit the property include potatoproteinase inhibitor II and soybean Bowman-Birk inhibitor, althoughother proteinase inhibitors with similar amino acid sequences and withsimilar proteinase inhibition properties may be used. While singleproteinase inhibitors have been shown to be effective, combinations oftwo or more distinct proteinase inhibitors may also be used.

In a preferred embodiment, a proteinase inhibitor product isolated frompotatoes is administered orally prior to a meal. The potato proteinaseinhibitor extract contains between about 15% and about 25% by weight PI2and also contains other proteins, including Bowman-Birk inhibitor. Thepotato proteinase inhibitor extract is present in an amount betweenabout 1 mg and about 1000 mg per dose, and preferably between about 5 mgand about 100 mg per dose, and most preferably between about 7.5 mg andabout 30 mg per dose. The potato proteinase inhibitor is effective toreduce the blood glucose spike following a meal by between about 5% andabout 30% and the AUC glucose by between about 5% and about 40%. Anotherpreferred proteinase inhibitor is Bowman-Birk inhibitor, which istypically isolated from soybeans. The Bowman-Birk inhibitor is presentin an amount between about 0.1 mg and about 5.0 mg per dose, andpreferably between about 0.5 mg and about 2.0 mg per dose. TheBowman-Birk inhibitor is effective to reduce the blood glucose spikefollowing a meal by between about 10% and about 25% and the AUC glucoseby between about 5% and about 30%.

It is an object of the present invention to reduce post-prandialglycemia in humans by the oral administration of one or more proteinaseinhibitors prior to a meal.

It is a further object of the invention to reduce the initial bloodglucose spike following a meal by the oral administration of one or moreproteinase inhibitors prior to the meal.

It is another object of the invention to reduce the total area under thecurve blood glucose following a meal by the oral administration of oneor more proteinase inhibitors prior to the meal.

It is yet a further object of the invention to treat hyperglycemia bythe oral administration of one or more proteinase inhibitors.

It is yet another object of the invention to prevent obesity by the oraladministration of one or more proteinase inhibitors.

Yet a further object of the invention is to combat Type II diabetesthrough the administration of one or more proteinase inhibitors eitheralone or in combination with other medications that are used incombating diabetes.

These and other objects of the invention will be understood by thoseskilled in the art upon a review of this specification, the associatedfigures and the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1 a and 1 b are HPLC chromatograms of the potato PI2 extract usedin the experiments and an authentic PI2 standard, respectively.

FIG. 2 is a photograph of an SDS PAGE of the potato PI2 extract used inthe experiments and an authentic PI2 standard.

FIG. 3 is a graph showing the effect of an increasing PI2 dose onpost-prandial integrated area under the blood glucose curve (AUC) aftera test meal.

FIG. 4 is a graph showing the effect of an increasing PI2 dose on theinitial rise in blood glucose above the baseline (A Glucose) thirtyminutes after a test meal.

FIG. 5 is a schematic diagram of the effects of chronic consumption of ahigh glycemic load.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

The composition for reducing post-prandial blood glucose levels inhumans is based on a proteinase inhibitor that delays gastric emptyingand reduces post-prandial glycemia which may be beneficial in combatingobesity and in the therapeutic treatment of patients suffering fromhyperglycemia. The proteinase inhibitor is believed to enhance therelease of cholecystokinin (CCK), a peptide which regulates gastricemptying. The preferred proteinase inhibitors include potato proteinaseinhibitor II and Bowman-Birk inhibitor. In particular, a proteinaseinhibitor extracted from potatoes and available commercially from KeminConsumer Care, L.C., Des Moines, Iowa, under the trademark Bioffect™ wasused in some of the examples. Bioffect™ is also available in tabletsformulated to contain 15 mg per dose and sold under the trademarkSatise™.

The invention is based on the surprising result that proteinaseinhibitors administered orally before a meal have the effect of reducingthe initial post-prandial glucose spike and also reduce the totalintegrated area under the curve blood glucose over more than three hoursafter a meal. Also surprising is that the proteinase inhibitors areeffective when administered in a dose in the less than ten milligramrange.

Example 1 Methods Subjects

Twenty-six men and 13 women, mean age 35 years (range 23-61 years) witha mean body mass index of 27 (range 23-32) participated in the study.Sample size was based on the study by Schwartz et al. who showedsignificant decreases in mean post-prandial glucose in six type IIdiabetic subjects following ingestion of a glucose/protein shake in thepresence and absence of a high dose of PI2 (1.5 g). All subjects gaveinformed consent before the study began, and could withdraw at any time.

Study Design

Subjects were randomly allocated to receive placebo and two of the threefollowing doses: 7.5, 15, or 30 mg PI2 extract. On each study daysubjects arrived at 8.00 AM after a 10 hour fast. They were givenbreakfast and 500 ml of water to drink throughout the morning, but atenothing further until the test meal. Height and weight of all subjectswere recorded during their first visit. Three and a half hours afterbreakfast the first blood glucose measurement was made, and subjectswere given the experimental capsule and 500 ml of water. Thirty minuteslater the test lunch was served. As soon as each subject completed themeal, the timing for post-prandial glucose measurements began. Subjectsrecorded any adverse reactions at fifteen minute intervals for 200minutes after eating the meal.

Test Meal

On each test day subjects were fed a breakfast of granola, skim milk,and orange juice that contained 330 kilocalories derived from 67 g ofcarbohydrate, 2.5 g of fat, and 12 g of protein. No other food waspermitted until the test meal, which was consumed at noon on the testday. The test meal was Chicken Teriyaki (Boston Market) and contained nopotato products. The nutritional content of the test meal is set out inTable 1. All subjects consumed all meals in their entirety.

TABLE 1 Characteristics of the test meal Lunch Test Meal Energy (kcal)460  Fat (g) 11 Carbohydrate (g) 53 Protein (g) 27 Number ofparticipants taking meal challenge after 39 placebo Average AUC forplacebo participants (SD) 2196.6 ± 1567.2

Glucose Measurements

Finger-prick capillary blood samples were taken 30 minutes before thetest meal (Baseline), and 30, 60, 90, and 120 minutes post-prandially.Glucose measurements were made with a Dex glucometer, Model #3952E(Bayer Pharmaceuticals), in accordance with the manufacturer'sinstructions.

Proteinase Inhibitor

PI2 extract was provided by Kemin Consumer Care, L.C. (Des Moines,Iowa), and was supplied in 00 gelatin capsules containing 7.5, 15, or 30mg, respectively. A mixture of dextrose and whey protein was used tobring all capsules to a uniform weight and volume and also served as aplacebo. The doses in the present study were chosen based on previousstudies demonstrating efficacy at 30 mg in liquid form (Spiegel, T. A.,Hubert, C. & Peikin, S. R. (1999) University of Medicine and Dentistryof New Jersey; Vasselli, J. R., Greenfield, D., Schwartz, L. &Heymsfield, S. B. (1999) Obesity Research Center, St. Luke's-RooseveltHospital Center, Columbia University), and 7.5 mg (Gary Green,University of Texas, San Antonio, 1996, 1997, unpublished data). Theactive material was produced from a single lot of potatoes (RussetNuggets; Kemin lot 87289C, approximately 244.39 mg PI2 extract/kg).

Measurement of PI2

RP-HPLC: Formulation of the active doses was based on quantitation byhigh performance liquid chromatography (HPLC). Reversed-phase HPLC(RP-HPLC) analyses were performed on a Hewlett Packard Model 1100equipped with a diode array detector using a Microsorb C-18, 5 μmparticle size, 300 Angstrom pore size, 4.6×250 mm (Varian AnalyticalInstruments, Walnut Creek, CA). The chromatographic conditions were asfollows: Isocratic elution for five minutes of 80% of 0.1% TFA in H₂O(20% of 1% TFA in acetonitrile). Gradient from 80-30% of 0.1% TFA in H₂O(20-70% of 1% TFA in acetonitrile) for 34 minutes. Gradient from 30-0%of 0.1% TFA in H₂O (30-100% of 1% TFA in acetonitrile) for 4 minutes.Flow rate was 1 ml/min for all gradients, and the column temperature wasmaintained at 30° C. Integration of the HPLC peak area provided therelative concentration of each sample (mg/g solids).

SDS-PAGE: To further characterize the PI2 extract, samples were analyzedby gel electrophoresis. SDS gels were prepared as 4% stacking, 15%resolving with 1.5 M Tris, 0.5 M Tris, 10% SDS, 30% ammonium persulfate,TEMED, and 40% Acryl/Bis. Wells were loaded with pre-stained marker, PI2standard, and PI2 extract. A current of 80 volts was applied for 1.5hours. Gels were then stained with Coomasie blue staining. Pure PI2standard was obtained by sequential RP-HPLC followed by gel filtrationchromatography. Western blot using a rabbit polyclonal antibodydeveloped by Kemin Foods, L.C. against PI2 protein, was used to furtherdetermine the identity of the major protein in the potato PI2 extractused in the current study.

Calculations and Statistical Analysis

The difference between the 30-minute post-prandial and baseline bloodglucose values was calculated for each subject visit (A glucose). Theintegrated area under the blood glucose-time curve (AUC) after each testmeal was calculated using the pre-meal value as the baseline, andintegrating the area from 0 to approximately 120 minutes after the meal.Repeated measure analysis of variance was used to test for significantdifferences between areas. The research design involved repeatedmeasures, so the PROC MIXED function in SAS was used, as this allows amore general specification of the covariance matrix of the dependentvariable, and allows random factors of both the model and error terms tobe correlated (Hongsen, Z. (2001) Proceedings of the 12th AnnualConference of the Midwest SAS Users Group, 132-140). All subjectsreceived placebo on one visit, but only two of the three possible activetreatments during the other visits, so an incomplete block design wasused to evaluate the relative effectiveness of the doses. The strategydescribed by Wolfinger (Wolfinger, R. D. (1993) Communications inStatistics, Simulation, and Computation 22, 1079-1106) was followed toselect an appropriate variance-covariance structure for the ANOVA test.The Akaike's Information Criterion was used to select the appropriatevariance-covariance structure for the model. Chi-square analysis wasused to evaluate data obtained as discrete variables with p<0.05considered to be significant.

Results

Doses of active PI2 extract were quantified by RP-HPLC. The integratedpeak representing the PI2 extract co-eluted with a pure authentic PI2standard, indicating that PI2 is contained in the extract and that it isthe major protein (FIGS. 1 a and 1 b). Results of gel electrophoresisfurther confirm the findings of the analysis by RP-HPLC and show thatthe PI2 in the extract is likely present as a monomer with a molecularweight of approximately ˜12 kDa (FIG. 2). MALDI MS analysis of thepurified PI2 protein demonstrated that this protein has a molecularweight of 12 kDa. Western blots of the separated proteins using a rabbitpolyclonal antibody for PI2 protein demonstrated that the major proteinband separated by SDS-PAGE is PI2. The actual amount of PI2 proteinpresent in a given extract could vary and ranges from 17-20%. The PI2extract was also characterized for its trypsin and chymotrypsininhibition activity using an in vitro assay demonstrating both trypsinand chymotrypsin inhibition. PI2 extract product contained a ratio of0.9-1.7:1 units of trypsin: chymotrypsin inhibition activity,respectively.

The volunteers in the present study consumed 120 test meals. Fortyplacebo doses were administered, along with 27 of each of the 7.5 mg and15 mg doses, and 26 of the 30 mg dose, respectively (one individualdeclined to provide blood samples and was included in determination ofadverse events monitoring). Table 1 shows the nutrient value of the testmeal and the mean glucose AUC following placebo. We first examined theeffect of PI2 extract on AUC; the repeated measure ANOVA model used forthis analysis showed a statistically significant effect of theexperimental treatment (f=3.3, p<0.05) but no statistically significantdifference between the experimental blocks. Subjects given a dose of 7.5mg PI2 extract before the test meal experienced no significant reductionin post-prandial glucose compared to placebo. The AUC of subjectsreceiving both 15 and 30 mg PI2 extract prior to the test meal wassignificantly reduced compared to placebo, but there was no significantdifference in post-prandial AUC between the two higher doses (FIG. 3).The decrease in AUC for 15 and 30 mg was 29.8% and 24.5% respectively,each compared to placebo. There was a significant reduction in glucoseat both the 15 mg and 30 mg dose levels compared to placebo, but therewas no significant difference in glucose between the two higher doses(FIG. 4). The decrease in glucose for the 15 and 30 mg doses was 25% and20% respectively, each compared to placebo.

Feeding 120 test meals resulted in 14 reports of an adverse reactionfrom subjects. These are summarized in Table 2.

TABLE 2 Subjects recording adverse effects after eating a test mealpreceded by PI2 extract Gastrointestinal Chi-square PI2 Extract Dose(mg) Symptoms Headache Total (vs. Placebo) 0 3 — 3 — 7.5 5 1 6 0.19 15 —2 2 1.76 30 3 — 3 0.23Gastrointestinal symptoms included nausea, cramping and diarrhea.Differences in occurrence rates of adverse reactions between thetreatments and the placebo were not significant (p>0.05, Chi square).Subjects experiencing symptoms rated them as mild, and frequently theywere noted at only one of the recording times.

Discussion

The drastic rise in obesity rates over the past 10 years has beenaccompanied by diets resulting in chronic glycemia and hyper secretionof insulin (Grundy; Wolever, T. M. & Bolognesi, C. (1996) J Nutr 126,2807-12). This, in turn, initiates a cascade of metabolic andphysiologic events resulting in decreased lipolysis, increased de novolipogenesis, and faster onset of hunger and subsequent food intake(Jenkins; Ludwig). Rapid and drastic excursions in blood sugar may notonly contribute to obesity but other chronic diseases, includingdiabetes and cardiovascular disease (as summarized in FIG. 5 and inLudwig).

Accordingly, lowering the glycemic load experienced by the body by dietor other means may be an effective way to reduce the post-prandialglycemia that can lead to weight gain and obesity. Findings of thepresent study suggest that it is possible to lower the glycemic loadexperienced by the body by ingesting a supplement containing a low doseof PI2 extract prior to a meal. Doses of either 15 mg or 30 mg taken 30minutes before a test meal significantly reduced the subsequent rise inblood glucose (FIGS. 3 and 4). A dose of 7.5 mg had no significanteffect, indicating that under these test conditions the lowest effectivedose lies between 7.5 and 15 mg. This study was limited to acuteobservations, and the effect of chronic oral administration of PI2extract on blood sugar levels remains to be studied. However, this studyis unique because PI2 has not previously been administered in solid formin an encapsulated supplement prior to the meal, and because a solidmixed meal was used for the first time. In addition, the dose used wassubstantially lower and less pure than that previously reported, and alarger cohort of subjects was studied. A dose of 1.5 g PI2 (90-100%pure) by column chromatography (Clarence Ryan, Washington StateUniversity, Pullman, Wash.), administered in liquid form was used in twoprevious studies; in one study PI2 was added to soup and fed 8 minutesbefore a test meal, and in the other it was incorporated in a testbeverage (Hill et al.; Schwartz et al.). In neither case was itencapsulated. Other differences include the size and glycemic index ofthe test meals and potential variations in the PI2 dose bioactivity. Wefound a mean reduction in post-prandial blood glucose AUC of 29.8% witha 15 mg dose of PI2 extract and 24.5% decrease with a 30 mg active dose(FIG. 3). Schwartz et al. reported a comparable 24.5% reduction in AUCafter feeding 1.5 g PI2 with a liquid glucose and protein beverageadministered to diabetics. While the dose of PI2 administered in thatstudy was apparently 100-fold larger, we cannot be sure that it was ofthe same specific activity as used in our current study. Therefore, itis unclear whether larger doses of PI2 extract would evoke a greaterresponse.

Inspection of the responses of individual subjects to placebo or activedose reveals that 9 subjects experienced no reduction in glycemia witheither of the two dose levels of PI2 extract administered(non-responders). There was no significant effect of BMI, age, orfasting blood glucose on responsiveness. Among the 9 non-responders fromthe initial study, 8 were male and one was female, although thisdifference was not significant (p=0.07, Chi-square).

The notion of lowering the glycemic load to reduce or maintain weight issupported by both animal and human studies. Normal rats fed isocaloricdiets differing dramatically in terms of glycemic load, experience largedifferences in post-prandial glycemia and insulin response (Kabir, M.,Rizkalla, S. W., Champ, M., Luo, J., Boillot, J., Bruzzo, F. & Slama, G.(1998) J Nutr 128, 35-43; Kabir, M., Rizkalla, S. W., Quignard-Boulange,A., Guerre-Millo, M., Boillot, J., Ardouin, B., Luo, J. & Slama, G.(1998) J Nutr 128, 1878-83). Maintaining rats on these diets for weeksat a time results in drastic differences in glucose and lipidmetabolism. The levels of fatty acid synthase and de novo lipogenesis,as well as adipocyte size, are higher in rats consuming a high vs. lowglycemic load diet (Kabir et al. 35-43; Kabir et al. 1878-83). Thesedata provide evidence at the cellular and metabolic level, that drasticelevations in blood sugar caused by exposing the body to a high glycemicload results in increased fat accumulation over a relatively shortwindow of time. Consistent with this are results from long term researchshowing that adult rats fed isocaloric diets evoking chronichyperglycemia gain a significant amount of weight while those fed a dietwith moderate glycemia maintain their weight (Pawlak, D. B., Denyer, G.S. & Brand-Miller, J. C. (2000) Proc Nutr Soc Aust 24, 215).

Weight loss studies in humans suggest that reducing the glycemiaexperienced by the body is an effective means to reduce and maintainweight. Subjects consuming an isocaloric diets consisting of lowglycemic index foods lose more weight or maintain their relative tothose consuming high glycemic index foods (Slabber, M., Barnard, H. C.,Kuyl, J. M., Dannhauser, A. & Schall, R. (1994) Am J Clin Nutr 60,48-53; Wolever, T. M., Jenkins, D. J., Vuksan, V., Jenkins, A. L., Wong,G. S. & Josse, R. G. (1992) Diabetes Care 15, 562-4; Clapp, J. R. (1997)Arch Gynecol Obstet 261, 101-107). These findings suggest thatmanipulation of the glycemic load, in these cases by consuming lowglycemic load diets, can effectively stimulate weight loss and/orprevent weight gain. Combined with the results from the present study,these support the hypothesis that PI2 extract can serve as an effectivenutraceutical to lower the glycemia experienced by the body, and mayhelp promote weight loss and reduce the propensity for weight gain.

PI2 extract is proposed to exert its effect on post-prandial glucose byenhancing the release of a well characterized peptide hormone, CCK,which is naturally secreted into the blood stream by enteroendocrinecells in response to a meal (Crawley, J. N. & Corwin, R. L. (1994)Peptides 15, 731-55). CCK acts on various target tissues throughout thebody including the gastrointestinal tract, where it delays gastricemptying leading to feelings of fullness, and the brain leading tofeelings of satiety. Although not measured in the present study,previous studies in the late 1980's and 1990's demonstrated that largedoses of purified PI2 enhance the release of CCK (Peikin et al.;Schwartz et al.) delay gastric emptying time (Schwartz et al.), anddecrease energy intake (Hill et al.) in humans. These were followed bystudies using a lower dose of less pure PI2 extract which demonstratedreduced hunger and increased fullness ratings (Spiegel et al.; Vasselliet al.) (summarized in Table 3). These findings are consistent with thewell established fact that PI's are potent stimulators of CCK release inrats (Liddle).

TABLE 3 Summary of PI2 Clinical Trials Study Institution Dose FormOutcome Spiegel et al. Columbia University  30 mg Liquid Significantdecrease in 1999 PI2 (pre-meal hunger ratings; increase extract shake)in fullness ratings; 2 kg weight loss Vasselli et al. Robert WoodJohnson  30 mg Liquid Significant decrease in 1999 Medical School, PI2(pre-meal hunger ratings; increase University of Medicine extract shake)in fullness ratings and Dentistry of New Jersey Schwartz et al.University of Texas, San 1500 mg Liquid Significant increase in 1994Antonio PI2 (shake) plasma CCK; delayed gastric emptying; decreasedblood sugar Hill et al. 1990 University of Leeds, U.K 1500 mg LiquidSignificant decrease in PI2 (pre-meal food consumption soup) Peikin etal. Robert Wood Johnson 1000 mg Liquid Significant increase in 1987Medical School, PI2 (shake) plasma CCK levels University of Medicine andDentistry of New Jersey Green, 1996-1997* University of Texas, San7.5-100 mg    Liquid Doses as low as 7.5 mg Antonio PI2 (shake) delayedgastric emptying and reduced blood sugar levels *Unpublished data

PI2 is a pH, heat, and salt stable protein (Bryant et al.), allowing itto be effective when administered orally, and making it unique amongplant PI's. The extract used in the present study contains PI2 (FIGS. 1and 2), and is derived from white potatoes using a method generally asdescribed in U.S. patent application Ser. No. 09/900,555, incorporatedherein by this reference. Although normally present in potatoes as adimer, the PI2 separated from our extract appears to be in the monomericform. The pure PI2 possess trypsin and chymotrypsin inhibitionactivities of 1.4 and 3.6 inhibition unites and the PI2 extract possessrelative trypsin and chymotrypsin inhibition activities of 22 and 13inhibition units. The added beneficial effect of reducing post-prandialglycemia makes PI2 extract a unique and promising nutraceutical.

Some studies involving the direct infusion of CCK have reported minoradverse side effects such as headache, nausea, and diarrhea (Crawley, J.N. & Corwin, R. L. (1994) Peptides 15, 731-55; Pi-Sunyer, X., Kissileff,H. R., Thornton, J. & Smith, G. P. (1982) Physiol Behav 29, 627-30). Forthis reason we questioned participants specifically about these effectswhich may ultimately have prompted reporting of events that wouldotherwise have gone un-noticed. Although there are a number of reportsin the literature demonstrating morphological changes in the pancreas asa result of long term exposure to extremely high doses of natural andsynthetic PI's in rodents, similar studies in pigs and primates are notassociated with such effects (Struthers, B. J., MacDonald, J. R.,Dahlgren, R. R. & Hopkins, D. T. (1983) J Nutr 113, 86-97; Harwood, J.P., Ausman, L. M., King, N. W., Sehgal, P. K., Nicolosi, R. J., Liener,I. E., Donatucci, D. & Tarcza, J. (1986) Adv Exp Med Biol 199, 223-37;Garthoff, L. H., Henderson, G. R., Sager, A. O., Sobotka, T. J., Gaines,D. W., O'Donnell, M. W., Jr., Chi, R., Chirtel, S. J., Barton, C. N.,Brown, L. H., Hines, F. A., Solomon, T., Turkleson, J., Berry, D., Dick,H., Wilson, F. & Khan, M. A. (2002) Food Chem Toxicol 40, 501-16;Garthoff, L. H., Henderson, G. R., Sager, A. O., Sobotka, T. J., O'Dell,R., Thorpe, C. W., Trotter, W. J., Bruce, V. R., Dallas, H. L., Poelma,P. L., Solomon, H. M., Bier, J. W., O'Donnell, M. W., Jr., Chi, R. K.,Chirtel, S. J., Barton, C. N., Brown, L. H., Frattali, V. P. & Khan, M.A. (2002) Food Chem Toxicol 40, 487-500). Such effects have yet to beobserved in humans using PI's from natural sources. Furthermore,previous studies using PI2 have not demonstrated any side effects withdoses many times that used in this study (Peikin et al.; Schwartz etal.). Side effects noted by our subjects were mild and inconsistent, andcaused no withdrawals from the study. No increasing dose response wasnoted for any of these effects and the rate of occurrence was notdifferent between placebo and treatment. If persistent use of PI2extract were contemplated we may see additional mild side effects,although it is equally possible that tolerance to undesired effectswould develop over time.

In conclusion, we have demonstrated in the largest randomized controlledclinical trial to date that a low dose of PI2 extract prior to astandardized meal reduces significantly post-prandial glycemia in themajority of healthy subjects. Additional studies will be required toascertain long term effects of this supplement on blood glucose,appetite and body weight. While a mechanism of action has been proposed,it will be important to confirm this hypothesis in future studiesaddressing changes in serum CCK, insulin, and the like. Such studiescould be instrumental in applying PI2 to the clinical problems ofobesity and diabetes.

Example 2

To better understand if the trypsin/chymotrypsin inhibiting activity ofthe PI2 protein was related to the glucose response to the potatoproteinase inhibitor extract, a preparation that purified the PI2fraction from the potato proteinase inhibitor extract (abbreviated pPI2)was tested along side a preparation of Bowman-Birk inhibitor after mealchallenge. The Bowman-Birk inhibitor used was obtained fromSigma-Aldritch and had a stated purity of greater than 80%. Bowman-Birkinhibitor has similar enzyme inhibiting properties as pPI2. The mealchallenge was conducted at a breakfast meal instead of a lunch meal, asin the prior study, and consisted of 390 kcal with 100 kcal from fat and53 g carbohydrate and was provided to individuals who had been fastingfor at least 10 hours. Each participant made two visits to the researchcenter and underwent two meal challenges—one for the placebo and one foran active (15 mg pPI2 or 0.8 mg Bowman-Birk inhibitor) and thesetreatments were provided in a double-blinded format. The randomizationscheme also prevented the participants or the study personnel fromknowing at which visit the placebo was given until the code was broken.

In this study we found that among healthy volunteers, both pPI2 andBowman-Birk inhibitor decreased the post-prandial glucose spike. Thedata is summarized in Table 4. For example G was decreased by 25.5%compared to placebo among the 10 individuals taking pPI2. Thisdifference (from a mean G of 52.1 mg/dl+15.9 sd for the placebo doseversus 38.8+34.6 sd for those taking the pPI2) was evaluated byone-tailed, paired student's t test and yielded p=0.065. The Bowman-Birkinhibitor also inhibited the post-prandial glucose spike with a decreasein G of 42.4%. The absolute decrease was from 47.9 mg/dl±22.6 sd for theplacebo treatments compared to 27.6 mg/dl±21.6 sd for the Bowman-Birkinhibitor treatment. This was significant by student's t test withp=0.04.

Neither pPI2 nor Bowman-Birk inhibitor showed statistically significantdifferences in AUC, although the trend was toward absolute decreases inthis parameter of 17% and 11.5% for both pPI2 and Bowman-Birk inhibitor,respectively.

In the earlier study we did discover that some individuals seemed to beunresponsive to pPI2. Again, we found this to be the case with pPI2.Three individuals were identified as non-responders with pPI2 and twoindividuals were unresponsive to Bowman-Birk inhibitor. A non-responderwas defined as an individual who did not have a lower absolute G aftertreatment with the active test material than with the placebo.

Thus far we are supporting the hypothesis that the PI2 protein in thepotato proteinase inhibitor extract and the trypsin/chymotrypsininhibiting activity may be related to modulation of post-prandialglucose since the relative concentration of this activity in the pPI2showed comparable glucose modulating activity as with the originalpotato proteinase inhibitor extract and the Bowman-Birk inhibitor whichalso contains an inhibitor or inhibitors of trypsin and chymotrypsinshowed glucose modulating activity.

TABLE 4 Comparison of purified PI2 and Bowman-Birk Inhibitor on G andAUC Purified PI2 Product Bowman-Birk Inhibitor Parameter Placebo ActivePlacebo Active Mean G ± Study 1 52.1 ± 15.9 38.8 ± 34.6 47.9 ± 22.6 27.6± 21.6 SD *p = 0.065 *p = 0.04 Mean G ± Study 2 55.1 ± 21.3 45.7 ± 21.749.8 ± 13.9   45 ± 21.8 SD *p = 0.058 *p > 0.05 Mean AUC ± Study 1 2279± 1187 2747 ± 1325 2015 ± 1702 1783 ± 1935 SD *p = 0.15 *p = 0.32 MeanAUC ± Study 2 2496.9 ± 1878.1 2153.6 ± 2181.3 SD *p = 0.229 *one tailedpaired t test (versus placebo)

Although the invention has been described with respect to a preferredembodiment thereof, it is to be also understood that it is not to be solimited since changes and modifications can be made therein which arewithin the full intended scope of this invention as defined by theappended claims.

We claim:
 1. A nutritional intervention composition for reducingpost-prandial blood glucose in humans, comprising between about 0.1 mgand about 500 mg of a proteinase inhibitor administered prior to themeal.
 2. A nutritional intervention composition as defined in claim 1wherein said composition is taken 1 to 30 minutes before the meal.
 3. Anutritional intervention composition as defined in claim 1, wherein theproteinase inhibitor is isolated from plant material.
 4. A nutritionalintervention composition as defined in claim 3, wherein the proteinaseinhibitor is derived from the group of plants consisting of potatoes,soy and beans.
 5. A nutritional intervention composition as defined inclaim 1, wherein the proteinase inhibitor is proteinase inhibitor IIisolated from potatoes.
 6. A nutritional intervention composition asdefined in claim 1, wherein the proteinase inhibitor is Bowman-Birkinhibitor isolated from soybeans.
 7. A nutritional interventioncomposition as defined in claim 1, wherein the proteinase inhibitorreduces the initial blood glucose spike.
 8. A nutritional interventioncomposition as defined in claim 1, wherein the proteinase inhibitorreduces the area under the curve blood glucose.
 9. A nutritionalintervention composition as defined in claim 8, wherein the area underthe curve blood glucose is measured over a time period of up to 4 hoursafter the meal.
 10. A nutritional intervention composition as defined inclaim 1, wherein the proteinase inhibitor reduces the initial bloodglucose spike by between about 5 percent and about 30 percent.
 11. Anutritional intervention composition as defined in claim 1, wherein theproteinase inhibitor reduces the area under the curve blood glucose overa period of about 3.5 hours following the meal by between about 5percent and about 40 percent.
 12. A nutritional intervention compositionas defined in claim 1, wherein the proteinase inhibitor is potatoproteinase inhibitor II administered orally in a dose of between about0.5 and about 500 mg.